Copyright Ó 2006 by the Genetics Society of America

DOI: 10.1534/genetics.105.049270

Effects of trans-acting Genetic Modifiers on Meiotic Recombination

Across the a1–sh2 Interval of Maize

Marna D. Yandeau-Nelson,*,†,1 Basil J. Nikolau‡ and Patrick S. Schnable*,†,§,**,2

*Interdepartmental Genetics Program, †Department of Genetics, Development and Cell Biology, ‡Department of Biochemistry, Biophysics
and Molecular Biology, §Department of Agronomy and **Center for Plant Genomics, Iowa State University, Ames, Iowa 50014-3467
Manuscript received August 8, 2005
Accepted for publication June 26, 2006

ABSTRACT
Meiotic recombination rates are potentially affected by cis- and trans-acting factors, i.e., genotype-
specific modifiers that do or do not reside in the recombining interval, respectively. Effects of trans
modifiers on recombination across the 140-kb maize a1–sh2 interval of chromosome 3L were studied in
the absence of polymorphic cis factors in three genetically diverse backgrounds into which a sequence-
identical a1–sh2 interval had been introgressed. Genetic distances across a1–sh2 varied twofold among
genetic backgrounds. Although the existence of regions exhibiting high and low rates of recombination
(hot and cold spots, respectively) was conserved across backgrounds, the absolute rates of recombination
in these sequence-identical regions differed significantly among backgrounds. In addition, an intergenic
hot spot had a higher rate of recombination as compared to the genome average rate of recombination in
one background and not in another. Recombination rates across two genetic intervals on chromosome 1
did not exhibit the same relationships among backgrounds as was observed in a1–sh2. This suggests that at
least some detected trans-acting factors do not equally affect recombination across the genome. This study
establishes that trans modifier(s) polymorphic among genetic backgrounds can increase and decrease
recombination in both genic and intergenic regions over relatively small genetic and physical intervals.

M EIOTIC recombination mediates the proper dis-

junction of chromosomes, generates novel allelic
combinations across chromosomes, and provides the
genetic diversity upon which selection can act. Accord-
ing to the double-strand break repair (DSBR) model
(Szostak et al. 1983; Sun et al. 1991), meiotic recom-
bination initiates with a double-strand break (DSB).
Fungal, animal, and plant chromosomes exhibit re-
gions of recombination hyperactivity, i.e., hot spots, and
hypoactivity, i.e., cold spots (reviewed in Lichten and
Goldman 1995; Schnable et al. 1998; Petes 2001;
Nachman 2002), as compared to the genomewide av-
erage rate of recombination in the organism being
studied. Consistent with the DSBR model, recombina-
tion hot spots in Saccharomyces cerevisiae are clearly asso-
ciated with DSBs (reviewed in Lichten and Goldman
1995) and these DSB hot spots are not distributed
randomly across the yeast genome (Gerton et al. 2000).
Nonrandom patterns of recombination have been
identified in all organisms studied to date. For example,
in most species recombination rates per megabase are
suppressed in and surrounding centromeres (Lambie
and Roeder 1986; Werner et al. 1992; Mahtani and
Willard 1998; Puechberty et al. 1999; Kagawa et al.
1
Present address: Pennsylvania State University, University Park, PA 16802.
2
Corresponding author: 2035B Roy J. Carver Co-Laboratory, Iowa State
University, Ames, IA 50014-3467. E-mail: schnable@iastate.edu
2002). Similarly, in most species, with the exception of
Caenorhabditis elegans, for which recombination rates
are negatively correlated with gene density (Barnes
et al. 1995), recombination rates are higher in gene-rich
than in gene-poor regions of the genome (reviewed in
Lichten and Goldman 1995; Schnable et al. 1998). In
wheat and barley, for example, analyses of integrated
genetic and cytogenetically based physical maps (Gill
et al. 1993, 1996a,b; Hohmann et al. 1994; Delaney et al.
1995a,b; Mickelson-Young et al. 1995; Kunzel et al.
2000) have shown that most recombination occurs in
relatively small gene-rich regions. Similarly, in maize
recombination nodules cluster in regions of high gene
density (Anderson et al. 2006). In addition, it is es-
timated that recombination in a gene-dense portion of
the bz1 region on chromosome 9S in maize is two orders
of magnitude greater than in a flanking region of high
retrotransposon density (Fu et al. 2002).
Although it is well established that high frequencies
of recombination are positively correlated with gene-
rich regions in the grasses, recombination breakpoints
within these hot spots might resolve equally in genic and
intergenic sequences. A high-resolution recombination
mapping study across the 140-kb multigenic maize
a1–sh2 interval supports that genes per se are preferred
recombination hot spots and intergenic regions are
cold spots; even so, nongenic hot spots and genic cold
spots do exist (Yao et al. 2002). These maize genic hot

Genetics 174: 101–112 (September 2006)

102 M. D. Yandeau-Nelson, B. J. Nikolau and P. S. Schnable
spots can have either nonrandom distributions of break-
points as observed in a1 (Xu et al. 1995) and other genes
(Eggleston et al. 1995; Patterson et al. 1995) or uni-
form distributions across the gene, as in bz1 (Dooner
and Martinez-Ferez 1997).
Variations in meiotic recombination rates occur not
only among various regions of the genome but also
among genetic backgrounds (reviewed in Simchen and
Stamberg 1969) as evidenced in both animals (Koehler
et al. 2002) and plants. In Arabidopsis, recombination
rates vary among accessions (Sanchez-Moran et al.
2002). In maize, heterogeneity in recombination fre-
quencies has been well documented within corn belt
(Beavis and Grant 1991; Tulsieram et al. 1992), corn
belt 3 exotic germplasm (Tulsieram et al. 1992), syn-
thetic (Fatmi et al. 1993), exotic, and teosinte-maize
hybrid (Williams et al. 1995) mapping populations and
has often confounded the generation of composite
genetic maps. Recombination rates in intervals with
heterogenous frequencies varied two- to threefold among
diverse mapping populations (Williams et al. 1995).
Within a mapping population, recombination frequen-
cies both in adjacent (Tulsieram et al. 1992) and be-
tween genetically unlinked (Beavis and Grant 1991;
Fatmi et al. 1993) regions were often not correlated,
demonstrating that recombination can be differentially
regulated across a chromosome. But in addition, several
intervals on different chromosomes were shown to have
either negatively or positively correlated recombination
frequencies (Fatmi et al. 1993). Together, these studies
suggest that the control of variation in recombination
rates might be polygenic and that some factors might
control specific genetic intervals.
The heterogeneity in recombination frequencies
among genetic backgrounds can be attributed to two
general classes of genetic factors: cis and trans. Cis-acting
elements are genetic factors that affect recombination
in the region in which the factor resides. A study of re-
combination across several teosinte a1–sh2 intervals,
each having large insertion/deletion polymorphisms
(IDPs) as compared to maize, has revealed significant
differences in recombination rates and distribution of
recombination breakpoints across the interval caused
by the action of cis factors (Yao and Schnable 2005).
Similarly, a not yet molecularly characterized cis factor
increases recombination in the large A188 Sh1–Bz1 in-
terval on maize chromosome 9S (Timmermanset al. 1997).
In contrast to cis elements, trans-acting factors are
genetic modifiers that are not closely linked to the in-
terval in which they affect recombination. Trans modi-
fiers might include factors involved in chromatin
remodeling, proteins involved in the recombination
machinery, and autonomous transposons. For example,
the rate of crossover (CO) increases near a Mu insertion
in a1 in the presence of MuDR transposase (Yandeau-
Nelson et al. 2005). Also, recombination is affected on
the maize chromosome 9 c1–sh1 and bz1–wx1 intervals
as well as chromosome 7 by a single unidentified trans
factor (Timmermans et al. 1997). The maize mapping
studies described earlier established recombination rate
heterogeneity among genetic backgrounds and suggest
that this variation is under polygenic control (i.e., mul-
tiple trans factors). However, these studies (Tulsieram
et al. 1992; Fatmi et al. 1993) were unable to distinguish
between the effects of cis- and trans-acting genetic modi-
fiers, both of which potentially influence recombination
rates. Also, available data only suggest that rates of re-
combination differ among genetic backgrounds, but
the manner in which factors can affect the distribution
of recombination breakpoints across a small physical
distance containing both genic and nongenic intervals
is unknown.
To elucidate the effects of trans-acting modifiers spe-
cifically, these factors must be studied in the absence of
polymorphic cis-acting effects. This can be achieved
by studying recombination across an interval that is
sequence identical in several different genetic back-
grounds. The 140-kb a1–sh2 interval on maize chro-
mosome 3L is an ideal system in which to study how trans
factors modulate both recombination rates and the dis-
tribution of recombination breakpoints across an in-
terval because (1) recombination events across the
interval are easily identified by nonparental kernel
phenotypes, (2) high-resolution mapping of recombi-
nation breakpoints is straightforward due to a signifi-
cant degree of sequence polymorphism between the
A1 Sh2 haplotypes and the availability of previously
developed IDP markers across much of the interval
(Xu et al. 1995; Yao et al. 2002), and (3) the interval is
multigenic (Yao et al. 2002), which allows for the study
of how trans-acting factors differentially affect recombi-
nation across genic and nongenic regions.
A sequence-identical a1Trdt sh2 interval that had been
introgressed into three unique genetic backgrounds
(maize inbreds A632, Oh43, and W64A) was used to
assess the extent to which trans-acting genetic modifiers
that were polymorphic among the backgrounds affect
meiotic recombination in the 0.1-cM a1–sh2 interval.
This study reveals that trans-acting factors can influence
both rates of recombination and distribution of re-
combination breakpoints within conserved hot and
cold spots and can convert an average spot to a hot
spot. The modifier(s) affecting recombination across
this small interval on chromosome 3L does not appear
to differentially affect recombination among genetic
backgrounds in the same way across an interval on
chromosome 1S, suggesting that at least some trans-
acting factors affect specific regions of the genome.
MATERIALS AND METHODS
Genetic stocks: The shrunken-2 (sh2) gene is located on
chromosome 3 0.1 cM centromere-distal to the a1 locus
(Civardi et al. 1994). Mutations at these loci condition
 Trans Modifiers of Recombination
shrunken and colorless kernel phenotypes, respectively. The
a1Trdt sh2 haplotype (GenBank accession no. AF072704) was
described previously (reviewed in Xu et al. 1995). The A1-LC
Sh2 haplotype (GenBank accession nos. AF434192, AF347696,
AF363390, X05068, and AF363391) is derived from the inbred
line C (LC), a color-converted version of W22.
The a1Trdt sh2 (A632)7, a1Trdt sh2 (Oh43)9, and a1Trdt sh2
(W64A)6 genetic stocks were gifts from David Glover (Purdue
University). To generate these stocks, the inbreds A632, Oh43,
and W64A were crossed by a line that was homozygous for
the a1Trdt sh2 haplotype. Using the a1 and sh2 mutants as
markers, the a1Trdt sh2 haplotype was backcrossed into the
inbreds for seven, nine, and six generations to generate the
trans stocks a1Trdt sh2 (A632)7, a1Trdt sh2 (Oh43)9, and
a1Trdt sh2 (W64A)6, respectively. The trans stocks were self-
pollinated to generate homozygous a1Trdt sh2 sources in each
of the three genetic backgrounds.
Isolation of genetic recombinants and estimations of
genetic distances: Recombinants across a1–sh2: To identify re-
combinants that resolve between a1 and sh2, each of the three
inbred trans stocks was used as a male onto the inbred line C
(cross 1). Recombination was measured in the three resulting
trans stock/line C F1’s, i.e., A632/LC, Oh43/LC, and W64A/
LC, via testcrosses (cross 2) as described by Yao et al. (2002)
and Yao and Schnable (2005). To control environmental
effects on recombination, these testcrosses were performed
during a single season (summer, 1997) in a single field.
Cross 1: A1-LC Sh2/A1-LC Sh2 (line C) 3 a1Trdt sh2/a1Trdt sh2
(trans stock)
Cross 2: A1-LC Sh2/a1Trdt sh2 (F1 from cross 1) 3 a1Trdt sh2/
a1Trdt sh2
Progeny from cross 2 segregated for parental colored round
and colorless shrunken kernels. Rare kernels with nonparen-
tal phenotypes (viz., colored shrunken and colorless round)
putatively carry recombination events between a1 and sh2.
Because the original A632, Oh43, and W64A inbreds are
recessive for r1, a gene that encodes a transcription factor that
acts upstream of a1 in anthocyanin biosynthesis, colorless
round (a19Sh2) kernels could be generated due to the ab-
sence of r1 and not because of resolution of a recombination
breakpoint between a1 and sh2. For this reason, only the
colored shrunken (A19sh2) recombinant class was analyzed in
this study. Putative A19sh2 recombinant kernels from each
inbred background were tested, confirmed, and made homo-
zygous as described in Xu et al. (1995). The actual number of
A19sh2 recombinants isolated from each genetic background
was estimated from the frequency of confirmed recombinants
out of the total number analyzed.
Recombinants across chromosome 1S: To identify genetic
intervals unlinked to the a1–sh2 interval on chromosome 3L,
1000 IDP genetic markers developed by the Maize Genetic
Mapping Project (http://maize-mapping.plantgenomics.iastate.
edu/) and genetically mapped to chromosomes 1–2, and 4–10
were surveyed. The criteria for selecting genetic intervals were
that: (1) they be defined by two IDP markers that were 1–20 cM
apart, (2) each IDP marker was polymorphic between line C
(the A1 Sh2 haplotype used in cross 1) and the three trans
stocks, and (3) the genetic interval could be assayed in each of
the three trans-stock experiments. Only two intervals that met
these criteria were identified; both were located on chromo-
some 1S. Interval 1S.1 is defined by genetic markers IDP194
and IDP254 and 1S.2 is defined by markers IDP112 and
IDP643. The IDP194 and IDP254 markers were designed from
MEST54-C06 (GenBank accession no. BM072826) and
MEST139-B10 (GenBank accession no. BM334583), respectively,
and map to positions 115.6 and 115.0 cM, respectively, on the
maize IDP_body map version 4 (http://magi.plantgenomics.
103
iastate.edu/cgi-bin/cmap). Markers IDP112 and IDP643 were
designed from MEST19-B03 (GenBank accession no. BG841229)
and MEST129-G08 (GenBank accession no. BM333984) and
map to positions 82.8 and 84.6 cM, respectively.
Mapping populations were generated by crossing a single F1
plant from cross 1 for each of the trans stocks to the inbred B77
(cross 3).
Cross 3: B77 3 A1-LC Sh2/a1Trdt sh2 (F1 from cross 1)
Consequently, the progeny of cross 3 potentially carry
alleles from the specific trans stock, line C, and B77. B77 was
selected as the female parent for cross 3 because when tested
with a sample of IDP markers across the genome it showed a
high frequency of polymorphisms relative to line C and each of
the three trans stocks (data not shown). Recombination rates
across 1S.1 and 1S.2 were assayed in populations of 700
seedlings derived from progeny of cross 3 in each genetic
background. Recombinants across each interval were identi-
fied as those individuals having nonparental combinations of
genotypes at the two linked loci in each interval. Genetic
distances in each background were calculated using the total
number of recombinants identified in each trans stock and the
total number of seedlings analyzed.
Genotypes were determined using PCR primers IDP112F,
59-CTGTGACATGTTTGATGCCC-39, IDP112R, 59-GGTGATG
ACCACGTACAAGC-39, and IDP112AW, 59-CCC TGC TGA
TAG TGA TAG AC-39; IDP643F, 59-ACCCTCATCTTCAGCAG
TCG-39 and IDP643R, 59-GGTGAAACGGCAGTACAAGG-39;
IDP194F, 59-GACAGATCCCTAACACTTGGG-39 and IDP194R,
59-AACAAGGCAACCTGTGAAGC-39; and IDP254F, 59-ATG
TTGGTTGAGCCTCTTGG-39 and IDP254R, 59-GATTCAGA
GAGAGTGCATGGC-39.
Seed sterilization, germination, and DNA isolation: Col-
ored shrunken kernels that were homozygous for recombi-
nant A19sh2 haplotypes originally isolated from cross 2 and
700 kernels from each genetic background derived from
cross 3 were germinated in 96-well flats. PCR-ready DNA was
isolated as described (Dietrich et al. 2002).
Due to fungal contamination, many shrunken kernels do
not germinate in the soil. For these recombinants, a second
aliquot of shrunken kernels was sterilized with pure bleach
(Clorox) for 1 min and then rinsed repeatedly (10–15 times)
with sterile water. Sterilized kernels were plated on moistened
autoclaved germination paper (Anchor Paper, St. Paul) in
sterile Petri dishes, treated with 0.5% Captan 50-W fungicide
(Platte Chemical, Fremont, NE), and covered with activated
charcoal. Seeds were germinated at 28° for 5–7 days. Seeds
were transferred to new plates, watered, and covered with fresh
charcoal every 1–2 days. Tissue was collected from coleoptiles
and DNA isolation was as described above.
Physically mapping recombination breakpoints: Recombi-
nation breakpoints across the a1–sh2 interval were mapped
relative to eight IDP markers that had previously been
identified between the A1-LC Sh2 and a1Trdt sh2 haplotypes
(YAO et al. 2002). These markers separated the a1–sh2 interval
into eight informative subintervals (Figure 1A); intervals are
numbered according to Yao et al. (2002). Allele-specific prim-
ers not described by Yao et al. (2002) are listed below. Primers
that when paired recognize a size polymorphism between the
two haplotypes are XL3, 59-ATGAGCGGGAGCCTATG-39, and
XL4, 59-TCAGCATCCATACCATTG-39. Allele-specific and size
polymorphism primers are shown in Figure 1A.
The breakpoints associated with .85% of the confirmed re-
combinants from the A632 (90%; 117/130), Oh43 (87%; 155/
178), and W64A (85%; 185/219) genetic backgrounds were
physically mapped. It was not possible to map the remainder
due to difficulties in obtaining homozygous recombinant
chromosomes or in germinating shrunken kernels.
104 M. D. Yandeau-Nelson, B. J. Nikolau and P. S. Schnable

TABLE 1
Genetic distances across a common a1–sh2 interval in three genetic backgrounds

Genetic No. No. No. No. No. correct Population Genetic

background isolateda recoveredb testedc confirmedd recombinantse sizes distances (cM)f
A632/LC 229 138 130 130 229 320,718 0.143 6 0.000667
Oh43/LC 323 188 188 178 306 758,271 0.081 6 0.00033
W64A/LC 442 219 219 219 442 704,077 0.126 6 0.000442
a
Number of putative colored shrunken recombinants isolated from cross 2 (materials and methods).
b
Number of putative recombinants successfully propagated.
c
Number of putative recombinants whose validity was tested (materials and methods).
d
Number of putative recombinants confirmed.
e
Number of correct recombinants ¼ no. isolated 3 (no. confirmed/no. tested).
f
Genetic distance ¼ 100 3 no. correct recombinants 3 2/population size. Because colorless round recombinants were not
isolated, genetic distances were calculated by multiplying the number of colored shrunken recombinants by two. Standard errors
were calculated according to the formula (pq/n)1/2. The genetic distances among genetic backgrounds varied significantly (P ,
0.03).

Statistical methods: For each physical interval examined,
genetic distances (in centimorgans) were compared among
genetic backgrounds using x2 homogeneity tests. For the
genetic distances between a1 and sh2, the corrected number
of A19sh2 recombinants (Table 1) was doubled because the
reciprocal class, a19Sh2, was not analyzed. This analysis makes
the reasonable assumption that the rates at which the two
recombinant classes arise are equal.
Statistical methods and population size adjustments were
performed as described by Yao and Schnable (2005). The
actual numbers of mapped recombinants and the adjusted
population sizes were used for subsequent x2 homogeneity
tests. Pearson x2 tests were conducted as described by Yao and
Schnable (2005) to compare the actual recombinant break-
point distribution in each genetic background across a1–sh2
to (1) the expected distribution if recombination across the
interval was equal to the genomewide average (2.4 cM/Mb)
and (2) the expected distribution of recombinants if the rate
of recombination across each subinterval was equal to the
average recombination rate across the entire a1–sh2 interval.
In this study, rates of recombination were compared to a
genomewide average recombination rate of 2.4 cM/Mb (Table
2). This is an updated rate compared to the 2.1 cM/Mb rate
(YAO et al. 2002) based on a more recent maize genetic map
that consists of 5917 cM (Lee et al. 2002).
The expected numbers of recombinants for each subinter-
val were calculated using the subinterval distances measured
in the line C haplotype (Figure 1A). The sizes of each
subinterval were previously determined via sequencing or
DNA gel blot analyses (Yao et al. 2002). For the a1Trdt sh2
haplotype, interval sizes were determined using the genomic
sequence from a1 to yz1 of this haplotype (GenBank accession
no. AF072704) for subintervals II (1.7 kb), III (1.34 kb), IV (0.4
kb), V (80 bp), VI (2.2 kb), VII (620 bp), and VIII (3.7 kb).
To test if the use of subinterval sizes from line C affects
comparisons of recombination rates to the genomewide and
to the a1–sh2 interval average rates of recombination, the
expected numbers of recombinants and the corresponding
statistical analyses were also conducted using subinterval sizes
from the a1Trdt sh2 haplotype. Because the significance or
nonsignificance for each subinterval was not altered by which
haplotype was used to determine sizes of each subinterval, only
statistical analyses using subinterval sizes measured in line C
are presented.
To investigate which subintervals contribute to the different
breakpoint distributions among the backgrounds, an explor-
atory statistical analysis was conducted. Freeman–Halton tests
as described in Yao and Schnable (2005) were conducted in
which each subinterval was successively removed (and then
replaced) from the analysis. If the recombination rates in a
subinterval were contributing to differences in breakpoint
distributions across the entire interval, then upon removal of
this subinterval (or group of subintervals) the backgrounds
should no longer differ significantly.
RESULTS
The genetic distance between a1 and sh2 and the
distribution of recombination breakpoints varies
among genetic backgrounds: A common a1Trdt sh2
interval was introgressed into the inbred lines A632,
Oh43, and W64A for multiple generations (materials
and methods). Each of the resulting trans stocks
contains a sequence-identical a1Trdt sh2 interval on
chromosome 3L, while the bulk of its genome is
expected to be derived from the recurrent parent: i.e.,
A632, Oh43, and W64A. These stocks are ideal for
studying the effects of trans-acting genetic modifiers on
recombination across the a1–sh2 interval because the
presence of an identical a1–sh2 interval in each of the
three backgrounds ensures that the cis factors that affect
recombination within the a1–sh2 interval are identical
in each of the inbred backgrounds. To analyze the
effects of genetic background on recombination across
the a1–sh2 interval, each of the trans stocks was crossed
to the inbred line C. Because each of the resulting F1’s is
identically heterozygous at the a1–sh2 interval (A1-LC
Sh2/a1Trdt sh2), the only modifiers of recombination
that are expected to differ among these three F1’s
should be outside of the a1–sh2 interval. Hence, any
observed differences in meiotic recombination across
the a1–sh2 interval among the three genetic back-
grounds can be attributed specifically to trans factors.
Meiotic recombination events that resolved between
a1 and sh2 and yielded colored shrunken kernels
(A19sh2) were identified from cross 2 (materials and
methods). Colorless round (a19Sh2) kernels were not
 Trans Modifiers of Recombination 105

TABLE 2
Statistical analyses of recombination across a1–sh2

Informative Genetic Comparisons to the Comparisons to the

subintervals backgrounds average of a1–sh2 a genome’s averagea Recombination activitiesb
II A632/LC 2.8e156[ (223) 4.2e63[ (103) Hot spot (global, local)
Oh43/LC 8.8e77[ (143) 5.9e13[ (3.63) Hot spot (global, local)
W64A/LC 3.5e100[ (153) 3.0e32[ (5.83) Hot spot (global, local)

III A632/LC 0.28 0.10 Average spot

Oh43/LC 0.73 0.094 Average spot
W64A/LC 0.41 0.41 Average spot

VI A632/LC 6.8e104[ (173) 2.1e40[ (7.53) Hot spot (global, local)

Oh43/LC 8.0e49[ (103) 6.3e07[ (2.63) Hot spot (global, local)
W64A/LC 0[ (233) 3.1e110[ (9.23) Hot spot (global, local)

VII A632/LC 7.1e6[ (6.93) 0.018[ (3.13) Hot spot (global, local)
Oh43/LC 0.16 0.55 Average spot
W64A/LC 0.030[ (3.33) 0.66 Hot spot (local)

VIII A632/LC 4.4e137[ (163) 3.3e52[ (7.13) Hot spot (global, local)
Oh43/LC 0[ (233) 9.7e81[ (5.83) Hot spot (global, local)
W64A/LC 4.9e175[ (143) 1.2e54[ (5.43) Hot spot (global, local)

IX A632/LC 3.1e8Y (NA)c 1.1e16Y (NA) Cold spot (global, local)

Oh43/LC 5.1e10Y (403) 1.5e36Y (1603) Cold spot (global, local)
W64A/LC 3.1e12Y (NA) 9.2e29Y (NA) Cold spot (global, local)

X A632/LC 0.74 0.23 Average spot

Oh43/LC 0.40 0.11 Average spot
W64A/LC 0.011 [(2.73) 0.89 Hot spot (local)

XI–XIII A632/LC 4.1e17Y (NA) 2.9e38Y (NA) Cold spot (global, local)
Oh43/LC 2.8e21Y (513) 2.1e86Y (2003) Cold spot (global, local)
W64A/LC 3.9e25Y (593) 1.6e66Y (1503) Cold spot (global, local)
a
P-values are shown for x2 goodness-of-fit tests used to compare the observed recombination rate in each subinterval with (a) the
average rate of recombination (cM/Mb) across the a1-sh2 interval in each genetic background and (b) the average rate of re-
combination in the genome (2.4 cM/Mb). This genome rate of 2.4 cM/Mb is an updated estimate based on a new maize genetic
map that consists of 5917 cM (Lee et al. 2002) compared to the rate previously used (Yao et al. 2002). Arrows ([ and Y) denote a
significantly higher or lower rate of recombination (P-value , 0.05), respectively, than the rate to which it was compared. For
subintervals with significant P-values, the fold difference between rates is shown in parentheses.
b
Hot and cold spots are intervals that have higher and lower rates of recombination, respectively, than the rates to which they are
compared. Global and local spots significantly differ from the average genome and the a1–sh2 interval rates of recombination,
respectively. Average spots do not significantly differ from either the global or local rates.
c
For genetic backgrounds that have no recombination in a given subinterval, the fold difference with the rate to which it is
compared cannot be calculated. NA, not applicable.

analyzed for reasons described in materials and
methods. Each of the genetic backgrounds exhibited
a statistically different rate of recombination across the
a1–sh2 interval and these rates varied almost twofold
(Table 1).
Rates of recombination across the a1–sh2 interval
differ significantly due to the action of trans-acting
modifier(s). To determine whether these trans-acting
factors also affect the distributions of meiotic recombi-
nation breakpoints across genic and nongenic regions
in this interval, recombination breakpoints isolated
from each genetic background were mapped relative
to sequence polymorphisms between the A1-LC Sh2
and a1Trdt sh2 haplotypes (materials and methods).
Because the a1–sh2 intervals present in each of the
genetic backgrounds were identical, breakpoints could
be mapped relative to the same sequence polymor-
phisms, thereby facilitating comparisons among genetic
backgrounds.
In each background recombination breakpoints were
not randomly distributed across the a1–sh2 interval
(P , 4.0e39). Indeed, .95% of the recombinants iso-
lated from each background mapped within the a1–yz1
interval (subintervals II–VIII; Figure 1, C and D) that
composes only 10% of the physical length of a1–sh2.
The majority of the remaining recombinants resolved at
106 M. D. Yandeau-Nelson, B. J. Nikolau and P. S. Schnable

Figure 1.—Distributions of recombination breakpoints across the a1–sh2 interval in the A632/LC, Oh43/LC, and W64A/LC genetic
backgrounds. (A) A schematic of the A1-LC Sh2 and a1Trdt sh2 haplotypes in which boxes represent genes. Subintervals are defined by
allele-specific PCR primers designed at sequence polymorphisms that distinguish the haplotypes and are numbered according to Yao et al.
(2002). Allele-specific primers are indicated by arrows above the haplotype they specifically amplify. The size of each subinterval in the line
C haplotype is listed. As compared to Yao et al. (2002), subintervals XI, XII, and XIII have been combined into a single interval (XI–XIII).
Because subintervals IV and V are very small and no recombinants mapped to these subintervals, they are considered not informative in
this study. (B) Rates of recombination in each subinterval. The * and ** denote significant differences relative to the genomewide average
recombination rate at the 0.05 and 0.01 levels, respectively. The rates of recombination in subintervals IX and XI–XIII are so low that they
are not visible on this graph. Subinterval X contains the 39 portion of x1. Even if all of the recombinants that resolved within subintervals
XI–XIII resolved within x1, the rate of recombination in x1 would still be significantly lower than the genomewide average in each of the
three backgrounds (P-value , 0.002). (C) The percentage of recombination breakpoints that mapped to each subinterval. (D) The num-
ber of recombination breakpoints that mapped to each subinterval. (C and D) In some cases, no breakpoints mapped to a subinterval in a
given genetic background and therefore no bar is shown. (B–D) Data from Yao et al. (2002) are provided for reference.
 Trans Modifiers of Recombination 107

TABLE 3
Statistical analysis of rates of recombination among genetic backgrounds

Comparisons among genetic backgroundsa

Subintervalsb A632/LC vs. Oh43/LC A632/LC vs. W64A/LC Oh43/LC vs. W64A/LC
II A . O (1.8e5) A . W (0.019) O ’ W (0.052)
III A ’ O (0.36) A ’ W (0.20) O ’ W (0.45)
VI A . O (2.6e5) A ’ W (0.36) W . O (5.4e9)
VII A . O (0.049) A ’ W (0.24) O ’ W (0.45)
VIII A ’ O (0.35) A ’ W (0.24) O ’ W (0.73)
IX A ’ O (0.51) A ’ W (ND)c O ’ W (0.38)
X A ’ O (0.83) A ’ W (0.24) O ’ W (0.16)
XI–XIII A ’ O (0.36) A ’ W (0.29) O ’ W (0.79)
a
For each subinterval, statistically significant relationships among rates of recombination in the A632/LC
(A), Oh43/LC (O), and W64A/LC (W) genetic backgrounds are shown. The P values associated with x2
homogeneity tests are provided in parenthesis. In a given subinterval, rates of recombination are indicated
as being not distinguishable (’) if P . 0.05.
b
The distribution of breakpoints across the entire a1–sh2 interval in the Oh43/LC background differed sig-
nificantly from the distributions in both the A632/LC (P ¼ 0.013) and W64A/LC (P ¼ 0.0008) backgrounds.
c
Because no recombinant breakpoints mapped to this interval in either background, rates of recombination
could not be compared. ND, not determined.

the 39 end of x1 (subinterval X; Figure 1, C and D). These
observations are consistent with previously described
distributions of recombination breakpoints across the
a1–sh2 interval (Yao et al. 2002; Yao and Schnable 2005).
The distributions of recombination breakpoints across
the eight a1–sh2 subintervals defined by sequence
polymorphisms between the A1-LC Sh2 and a1Trdt sh2
haplotypes (Figure 1) were compared among genetic
backgrounds (materials and methods). The distribu-
tion of breakpoints in the Oh43/LC background dif-
fered significantly from the distributions in both the
A632/LC and W64A/LC backgrounds (Table 3). This
finding indicates that trans-acting factors that are poly-
morphic in the Oh43/LC background as compared
to the other two genetic backgrounds affect not only
rates of recombination across the entire a1–sh2 interval
but also the distribution of recombination breakpoints
within this interval. Using the exploratory statistical
analysis described in materials and methods it was
possible to establish that in the Oh43/LC background
the interloop (subinterval VI) and the yz1 gene (sub-
interval VIII) were the most significant contributors to
the differences in the distribution of breakpoints across
the a1–sh2 interval.
Recombination hot and cold spots are mostly
conserved among genetic backgrounds: As defined by
Yao and Schnable (2005) ‘‘global’’ recombination hot
and cold spots are regions that exhibit rates of recom-
bination that are either higher or lower, respectively,
than the genomewide average rate of recombina-
tion (2.4 cM/Mb). ‘‘Local’’ recombination hot and cold
spots are regions that have rates of recombination that
are higher and lower, respectively, than the average rate
of recombination across the entire a1–sh2 interval in the
genetic background being examined. In each of the
genetic backgrounds examined in this study the average
recombination rate across the a1–sh2 interval (i.e., the
local average) is lower than the genomewide average
rate of recombination. ‘‘Average’’ spots exhibit rates of
recombination that do not differ significantly from the
genomewide average.
All three recombination hot spots previously identi-
fied by Yao et al. (2002) in the same A1-LC Sh2/a1Trdt
sh2 heterozygote used in this study but in another
genetic background [the a1 gene (subinterval II), the
yz1 gene (subinterval VIII), and the apparently non-
genic interloop (subinterval VI)] are both global and
local recombination hot spots in all three genetic
backgrounds (Figure 1B; Table 2). Similarly, two inter-
genic regions and one gene (x1) identified by Yao et al.
(2002) as recombination cold spots (subintervals IX, X,
and XI–XIII, Figure 1B; Table 2), are also cold spots in
all three genetic backgrounds analyzed in this study.
Trans-acting factors affect rates of recombination
within hot spots: As discussed above, the a1 (subinterval
II) and yz1 (subinterval VIII) genes and the nongenic
interloop (subinterval VI) are conserved global recom-
bination hot spots in each of the three genetic back-
grounds (Table 2). If trans-acting modifiers affect each
subinterval equally, the rates of recombination in each
subinterval should be highest and lowest in the genetic
backgrounds that have the largest (i.e., A632/LC) and
smallest genetic distances (i.e., Oh43/LC), respectively,
across the entire a1–sh2 interval (Table 1). Because this
is often not true (Figure 1; Table 3), we conclude that
trans-acting factor(s) that are polymorphic among the
genetic backgrounds are differentially affecting rates of
recombination within the conserved hot spots.
The most notable example of differential control of
recombination among genetic backgrounds occurs in
108 M. D. Yandeau-Nelson, B. J. Nikolau and P. S. Schnable

TABLE 4
Genetic distances across two genetic intervals unlinked to a1–sh2 in three genetic backgrounds

1S.1 interval 1S.2 interval

Genetic No. Population Genetic No. Population Genetic
background recombinantsa sizes distances (cM) recombinantsa sizes distances (cM)
A632/LC 15 704 2.13 6 0.06b 5 729 0.69 6 0.03c
Oh43/LC 15 717 2.09 6 0.05 1 705 0.14 6 0.01
W64A/LC 6 678 0.89 6 0.04b 0 678 —c
a
Total number from the two expected classes of recombinants.
b
The 2.4-fold larger genetic distance in A632 as compared to W64A is weakly supported (P-value ¼ 0.058).
c
The genetic distance in A632 is higher than in W64A (P-value ¼ 0.03).

yz1 (subinterval VIII; Figure 1A). Among the three
backgrounds, the rate of recombination across a1–sh2
is lowest in Oh43/LC (0.607 cM/Mb) and consistent
with this low rate most subintervals show the lowest rate
of recombination in this background (Figure 1B).
However, more than half the recombination events
isolated in the Oh43/LC background resolve in yz1
(Figure 1, C and D) and the corresponding rate of recom-
bination is 23-fold higher than the rate across a1–sh2
(P-value 0; Table 2) in this background, making yz1
the most recombinationally active region in Oh43/LC.
In contrast, only 40% of the breakpoints associated
with recombinants isolated in the A632/LC and W64A/
LC genetic backgrounds resolve within yz1 (Figure
1C). The recombination rate in yz1 is approximately
seven-, approximately six-, and approximately fivefold
hotter than the genomewide rate (Table 2) in the A632/
LC, Oh43/LC, and W64A/LC genetic backgrounds,
respectively, with no significant differences in recombi-
nation rates among the backgrounds (Figure 1B; Table
3). In each of the backgrounds, the rate of recombina-
tion in each of the subintervals of yz1 did not differ
significantly from the rate of recombination for the
entire gene (P-value . 0.05) and, further, the recombi-
nation rates in each of the subintervals of yz1 did not
differ significantly among the backgrounds (data not
shown).
Trans-acting modifiers can convert a region with an
average recombination rate into a hot spot: Although
their levels of recombination activity vary, recombina-
tion hot and cold spots are generally conserved among
the three genetic backgrounds. The exception, how-
ever, is subinterval VII, a nongenic region proximal to
yz1. While no recombination was observed in this in-
terval in another genetic background (Yao et al. 2002),
in the A632/LC genetic background subinterval VII is
approximately threefold more recombinationally active
than the genomewide average (7.4 cM/Mb; P-value ¼
0.018; Table 2; Figure 1B) and approximately sevenfold
hotter than the average rate across a1–sh2 in the A632/
LC background (P-value ¼ 7.1e06; Table 2). In the
W64A/LC background, however, subinterval VII is an
average spot when compared to the genomewide
average, but locally it is approximately three times more
recombinationally active than the average rate across
a1–sh2 (P-value ¼ 0.03; Table 2). In Oh43/LC, sub-
interval VII is an average spot both globally and locally
(Figure 1B; Table 2). In summary, this interval is more
recombinationally active in each of the genetic back-
grounds in this study compared to the sequence-identical
interval in the line C background (Yao et al. 2002).
Genetic distances of chromosomal intervals on
chromosome 1: The genetic distances between a1 and
sh2 differ significantly among the three genetic back-
grounds tested in this study. To determine whether
trans-acting modifiers that affect recombination across
the a1–sh2 interval of chromosome 3 are global or
region-specific modifiers of recombination, recombina-
tion was assayed in 700 progeny of cross 3 in two
intervals on chromosome 1 (intervals 1S.1 and 1S.2) in
the three genetic backgrounds. Intervals 1S.1 and 1S.2
are separated by 30.4 cM on chromosome 1S in the
maize IBM mapping population (IBM_IDP1MMP_
body map version 4.0; http://magi.plantgenomics.iastate.
edu/cgi-bin/cmap). In both intervals, the genetic dis-
tances were highest in the A632/LC background
consistent with results in a1–sh2 and, unlike in the
a1–sh2 interval, lowest in W64A/LC (Table 4). There-
fore, the relationships between genetic distances among
the genetic backgrounds differ between the intervals
examined on chromosome 1S and the a1–sh2 interval
on 3L.
DISCUSSION
Trans-acting genetic modifiers affect rates of recom-
bination in the a1–sh2 interval: The heterogeneity
in rates of recombination observed in previous studies
among maize genomes (Beavis and Grant 1991;
Tulsieram et al. 1992; Fatmi et al. 1993; Williams et al.
1995) could be due to cis- and/or trans-acting genetic
modifiers. In contrast, the genetic stocks used in this
study ensured that elements that affect recombination
in cis are the same in each background. This is, there-
fore, the first molecular analysis in plants, animals, or
 Trans Modifiers of Recombination
yeast for which differences in recombination across a
multigenic interval can be directly attributed to trans-
acting modifiers that differ among genetic backgrounds.
To enhance our ability to identify inbred back-
grounds that exhibit polymorphic trans-acting factors,
recombination was assayed in three genetically distinct
maize inbreds. A632 is a Stiff Stalk (SS) Reid Yellow Dent
inbred, while the Non-Stiff Stalk (NSS) inbreds Oh43
and W64A (http://www.maizegenetics.net/index.php?
page¼germplasm/lines.html) are members of the
Lancaster Sure Crop and the Hy:T8:Wf9 subgrouping
(Liu et al. 2003), respectively. A632 and Oh43 are among
the six inbreds that contribute to 70% of all hybrids in
the United States (Nass and Paterniani 2000).
Rates of recombination between a1 and sh2 varied up
to approximately twofold among these three genetic
backgrounds (Table 1). This establishes that trans-acting
modifiers can affect rates of recombination and, not
surprisingly, the effects of these factors vary among the
inbreds within different heterotic groups.
Trans effects on a multigenic interval: Unlike pre-
vious studies of trans-acting effects that used large
genetic (and physical) intervals with uncharacterized
molecular structures (Timmermanset al. 1997) or studies
that compare genetic sizes among different genetic
maps (Beavis and Grant 1991; Tulsieram et al. 1992;
Fatmi et al. 1993; Williams et al. 1995), analysis of the
well-characterized multigenic a1–sh2 interval (Figure
1A) allowed for observation of the effects of trans-acting
modifiers in defined genic and intergenic regions.
Although recombination hot and cold spots are
largely conserved among the genetic backgrounds
(Figure 1B; Table 2), the rates within recombination
hot spots are affected by trans-acting factors that are
polymorphic among the backgrounds (Table 3). For
example, in the Oh43/LC genetic background trans-
acting factor(s) have seemingly redirected recombina-
tion events that might normally resolve within the
nongenic interloop (subinterval VI) to yz1 (subinterval
VIII) such that the resulting rate of recombination in yz1
does not differ significantly from the rates in the A632/
LC and W64A/LC genetic backgrounds. In each of the
conserved recombination hot spots, the relationships
between recombination rates among the backgrounds
vary (Table 3). This demonstrates that the trans-acting
modifier(s) act by increasing or decreasing the rates of
recombination within particular hot spots.
Trans-acting modifier(s) affecting recombination in
this study are affecting not only the overall rate of re-
combination between a1 and sh2 but also the distribu-
tions of recombinants across this interval. This observation
is in direct contrast to the trans-acting autonomous
transposon MuDR that increased the rate of recombi-
nation at an a1 allele containing a Mu1 insertion but
did not change the distribution of recombinant break-
points compared to patterns observed in other a1 al-
leles (Yandeau-Nelson et al. 2005). This suggests, not
109
surprisingly, that different types of trans-acting modi-
fiers will affect recombination in different ways.
Trans-acting modifier(s) can act as hot and cold
switches in a region of lower sequence similarity: Al-
though there are exceptions (Yao et al. 2002), in general
plant genes are recombination hot spots and nongenic
regions are cold spots (reviewed in Puchta and Hohn
1996; Schnable et al. 1998). Indeed, nongenic sub-
intervals IX and XI–XIII are both local and global cold
spots (Figure 1B; Table 2) in each of the three genetic
backgrounds. In striking contrast, the nongenic sub-
interval VII is both a global and local hot spot in A632/
LC, providing a second example of an apparently
nongenic recombination hot spot. Because subinterval
VII is only a local hot spot in the W64A/LC and an
average spot in the Oh43/LC genetic backgrounds
(Table 2), trans-acting modifiers not only are able to
alter rates of recombination within hot spots but also
can switch hot spots on and off.
Recombination hot spots have been shown to be
‘‘dialed down’’ by sequence heterology (cis-acting ele-
ments) in many organisms, including within the maize
a1–sh2 interval (Yao and Schnable 2005), by a mech-
anism involving mismatch repair proteins (reviewed in
Modrich and Lahue 1996; Borts et al. 2000; Evans
and Alani 2000; Schofield and Hsieh 2003). At the
maize bz1 locus, as little as 1.5% sequence divergence
reduces rates of recombination twofold (Dooner
2002). In subinterval VII, the level of sequence heterol-
ogy between the line C haplotype and the a1Trdt-sh2
haplotype present in each genetic background is 3.1%.
Even with this relatively high degree of heterology, the
rate of recombination in A632 is approximately three-
fold higher than the average rate of recombination in
the genome (7.4 vs. 2.4 cM/Mb; Table 2). This demon-
strates that trans-acting modifiers in the A632/LC and
W64A/LC (where recombination in this subinterval is
also increased but to a lesser extent) backgrounds can
overcome the suppression of recombination that often
occurs between heterologous sequences. Further, this
establishes that high sequence identity, a cis-acting
factor, is not the only criterion that dictates whether a
region is a recombination hot or cold spot.
Comparison of cis- and trans-acting effects on
recombination: Both cis- and trans-acting factors can
potentially affect the rate of recombination in a given
interval. Only by studying each type of factor in the
absence of the other (e.g., trans in the absence of
polymorphic cis effects) is it possible to assess how these
two categories similarly and differently affect recombi-
nation. To that end, the a1–sh2 interval in maize is the
first region for which the effects of trans-acting modi-
fiers on recombination (this study) can be compared to
the effects of cis-acting elements on recombination that
were characterized among different a1–sh2 teosinte
intervals introgressed into maize (Yao and Schnable
2005).
110 M. D. Yandeau-Nelson, B. J. Nikolau and P. S. Schnable
Both the cis- and trans-acting modifiers in the two
studies affect recombination similarly in that in both
studies most of the recombination breakpoints resolve
in the proximal 10% of the a1–sh2 interval. Together
these studies suggest that although cis and trans effects
on recombination in the a1–sh2 interval are, in general,
similar, the cis elements represented in the teosinte a1–
sh2 intervals affect recombination to a greater extent
than do the trans-acting modifiers that are polymorphic
in the A632/LC, Oh43/LC, and W64A/LC genetic
backgrounds. For example, in a subinterval containing
the interloop (subinterval VI in this study), which is a
recombination hot spot in each of the trans stocks, cis
elements act to make this region an average, cold, and
hot spot in the three different intervals studied. Such
comparisons are limited, however, because the trans-
acting modifiers in the cis studies most likely differ from
those in the inbred backgrounds in this study.
Do the trans-acting factors act globally or on specific
genetic intervals? Trans-acting modifiers of meiotic
recombination (reviewed in Wahls 1998) have been
divided into two categories: those that affect recombi-
nation across the entire genome (i.e., global modifiers)
and those that act only on specific (i.e., region-specific
modifiers) genetic intervals (Catcheside 1977). Global
trans-acting modifiers are most likely proteins intimately
involved in the mechanisms of recombination (re-
viewed in van den Bosch et al. 2002). For example, in
yeast rates of meiotic CO are globally reduced in
mutants of DNA polymerase d (Maloisel et al. 2004).
In maize, desynaptic (Ji et al. 1999), which is involved in
crossover control, affects recombination across the
genome (Bass et al. 2003).
To test whether or not the trans-acting factors that
affect the a1–sh2 interval are global modifiers, in each
genetic background recombination was assayed in two
intervals genetically unlinked to a1–sh2. In both inter-
vals on chromosome 1, the relationships among genetic
distances were the same. However, these relationships
differed from that seen in the a1–sh2 interval (compare
Tables 1 and 4). This could be due to the same trans-
acting modifier(s) within a genetic background differ-
entially affecting recombination in chromosomes 1S
and 3L. Alternatively, different region-specific modifiers
could be acting on at least portions of each of the two
chromosomes. In addition, because the 1S.1 and 1S.2
intervals are most likely not sequence-identical among
the backgrounds we cannot rule out the possibility that a
combination of both trans- and cis-acting elements is
affecting recombination in these intervals. Hence, these
results suggest, but do not prove, that the trans-acting
modifiers detected in this study do not have global
effects and are instead region specific.
Region-specific trans-acting modifiers have been char-
acterized in Schizosaccharomyces pombe (De Veaux et al.
1992; De Veaux and Smith 1994; Li et al. 1997;
Krawchuk et al. 1999; Pryce et al. 2005) and Saccharo-
myces cerevisiae (Rockmill and Roeder 1990). The
effects of chromatin organization on DSB formation and
subsequent meiotic recombination repair have been
extensively demonstrated in many organisms (reviewed
in Lichten 2001; Petes 2001), including plant species
(reviewed in Schuermann et al. 2005). In S. pombe, rec10,
a protein involved in the formation of lateral elements,
is required for the activation of some but not all M26-
containing recombination hot spots (Pryce et al. 2005).
In this case, higher-order chromatin structure affects
recombination at a known hot spot. Trans-acting modi-
fiers involved in chromatin organization or remodeling
might be responsible for the transformation of the
nongenic subinterval VII from an average spot in
Oh43/LC to a hot spot in the A632/LC genetic
background by opening the chromatin and allowing
access by the recombination machinery. If so, trans-
acting modifiers of chromatin most likely act in context
of local structures (e.g., sequence motifs). Alternatively,
genotype-specific patterns of crossover interference
(reviewed in van Veen and Hawley 2003; Hillers
2004; Copenhaver 2005) could differently affect the
distributions of recombination hot spots across a
chromosome among genetic backgrounds.
Region-specific trans-acting modifiers could also in-
clude proteins (e.g., transcription factors) for which
binding to specific cis-acting elements [e.g., unique
sequence motifs (Wahls and Smith 1994; Kon et al.
1997; Pryce et al. 2005) or promoters] is necessary for
activation of recombination hot spots as seen in a-hot
spots in yeast (reviewed in Lichten and Goldman
1995; Petes 2001). Such interactions between cis-acting
elements and trans-acting modifiers, if they occur in
a1–sh2, must be due to differences among the trans-
acting modifiers (e.g., binding specificity or affinity
for the cis element) because the a1–sh2 intervals are
sequence-identical among the genetic backgrounds.
We thank Dan Nettleton (Iowa State University) for advice re-
garding statistical analyses, Heather Smith for the isolation and
purification of recombinant alleles, undergraduate students Brian
Reinertson, Timothy Dunham, John Tenhunfeld, and Lakeysha
Toomer for technical assistance, and David Glover (Purdue Univer-
sity) for the gift of the trans stocks. This research was supported in part
by the National Research Initiative of the United States Department of
Agriculture Cooperative State Research, Education and Extension
Service, grant numbers 9701407 and 9901579 to P.S.S. and B.J.N.
and 0101869 and 0300940 to P.S.S., and supported by Hatch Act and
State of Iowa funds.
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Communicating editor: A. H. Paterson